DESCRIPTION: Recombinant adenovirus is one of the most promising vectors for gene therapy of genetic and metabolic diseases due to its highly efficient gene delivery. Recent studies have shown that the success of adenovirus-mediated gene therapy is limited by two major drawbacks, inflammatory reactions elicited by low levels of viral gene expression in transduced cells and the short duration of therapeutic gene expression. The major goal of this proposal is to develop a hybrid adeno-EBV vector system that would provide persistent gene expression without immunogenicity in transduced cells. The vector is a complex form of a deltavector (or gutless vector). It harbors an EBV replicon that can be launched within the target cells in a stable circular form, and it is devoid of all adenoviral genes to avoid cytotoxicity T-lymphocyte (CTL) responses. Our efforts have been focused on developing the four essential components of the vector: 1) a CRE-loxP-independent packaging system to produce high-titer delta-vectors, 2) a cloning strategy to construct large linear delta-vectors, 3) a linear EBV vector that can be recircularized from the delta-vector, and 4) a molecular switch to regulate the release of the EBV vector from the adenovirus construct. We have successfully constructed and tested all four components, and we have developed a highly efficient packaging system to produce gutless-adenovirus vectors. We will continue our research to combine these components into a single vector system that can introduce therapeutic genes into target cells without induction of CTL response by the host. We will also characterize these hybrid vectors in cell culture and in animal models. We will develop additional helper viruses with different serotypes to test the feasibility of repeated gene transfer and expanding the vector tropism by rotating the serotypes of the helper viruses. When we achieve our goals, we will have a vector system that can efficiently transfer gene in vivo, that can provide persistent gene expression, that does not induce CTLs, that can be repeatedly administered, and that has an expanded tissue tropism.